ABCC9 is a novel Brugada and early repolarization syndrome susceptibility gene

Abstract

Background: Genetic defects in KCNJ8, encoding the Kir6.1 subunit of the ATP-sensitive K(+) channel (I(K-ATP)), have previously been associated with early repolarization (ERS) and Brugada (BrS) syndromes. Here we test the hypothesis that genetic variants in ABCC9, encoding the ATP-binding cassette transporter of IK-ATP (SUR2A), are also associated with both BrS and ERS.

Methods and results: Direct sequencing of all ERS/BrS susceptibility genes was performed on 150 probands and family members. Whole-cell and inside-out patch-clamp methods were used to characterize mutant channels expressed in TSA201-cells. Eight ABCC9 mutations were uncovered in 11 male BrS probands. Four probands, diagnosed with ERS, carried a highly-conserved mutation, V734I-ABCC9. Functional expression of the V734I variant yielded a Mg-ATP IC₅₀ that was 5-fold that of wild-type (WT). An 18-y/o male with global ERS inherited an SCN5A-E1784K mutation from his mother, who displayed long QT intervals, and S1402C-ABCC9 mutation from his father, who displayed an ER pattern. ABCC9-S1402C likewise caused a gain of function of IK-ATP with a shift of ATP IC₅₀ from 8.5 ± 2 mM to 13.4 ± 5 μM (p<0.05). The SCN5A mutation reduced peak INa to 39% of WT (p<0.01), shifted steady-state inactivation by -18.0 mV (p<0.01) and increased late I(Na) from 0.14% to 2.01% of peak I(Na) (p<0.01).

Conclusion: Our study is the first to identify ABCC9 as a susceptibility gene for ERS and BrS. Our findings also suggest that a gain-of-function in I(K-ATP) when coupled with a loss-of-function in SCN5A may underlie type 3 ERS, which is associated with a severe arrhythmic phenotype.

Keywords: ATP-sensitive potassium channel; J wave syndromes; Mutation; Sodium channel; Sudden cardiac death.

Figure 1. Electrocardiograms (ECGs) of ABCC9-V734I variant carriers (Proband 4, 5 and 6 in Table 2)

A and B: The ECG of first and second proband both showed sinus bradycardia and typical global J wave elevation as arrows indicated. C-1: ECG of third proband displayed 1^st degree atrioventricular block, ventricular bigeminy and an early repolarization pattern including prominent distinct J waves. C-2: Ventricular tachycardia/ventricular fibrillation (VT/VF) in the proband was precipitated by a closely coupled premature beat. Polymorphic VT degenerated into VF and was terminated by an appropriate implantable cardioverter defibrillator discharge. Arrows denote prominent J waves.

Figure 2. ABCC9-V734I mutation causes a gain of function in ATP-sensitive potassium channel (K_ATP) activity by reducing sensitivity of K_ATP channels to ATP

A: Chromatogram showing a heterozygous A insertion at nucleotide 3891 (exon 22) of SCN5A, predicting G1297G FSX22, a stop codon and truncation of Na_v1.5 protein. B: Chromatogram showing a heterozygous G-to-A transition at nucleotide 2200 (exon 17) in ABCC9, predicting a substitution of Isoleucine (I) for Valine (V) at residue 734 (V734I). C: Amino acid alignment performed using GenBank accession numbers corresponding to protein sequences shows that a Valine at position 734 is highly conserved among species. D: Sensitivity to ATP of KCNJ11-wild type (WT)/ABCC9-WT and KCNJ11-WT/ABCC9-V734I channels measured using an excised inside-out patch. E: Graph depicting IC₅₀ for Mg-ATP inhibition of I_K-ATP for WT- and V734I-ABCC9 channels. Mean ± SEM. **p<0.01 vs. WT.

Figure 3. Pedigree and representative electrocardiogram (ECG) tracings of proband 1 and affected family members

A: The family pedigree of BS094. Circles represent female subjects and squares represent male subjects. The arrow denotes the proband. −/− wild type (WT); +/− heterozygous for the mutation. Brugada, long QT, early repolarization (ER) syndromes, sinoatrial block are labeled by black, upward diagonal, downward diagonal, and dark grey symbol; unaffected subject is shown as white. B: 12-lead ECG of proband’s father (I-1) showing accentuation of ER pattern (arrows) in lead II, III, aVF and V₆. C: ECG of the proband’s mother before and after ajmaline (I-2); QT and QTc intervals are prolonged. D: ECG of proband’s younger brother (II-2) is normal. E: ECG of proband-1 recorded before and after ajmaline (II-1); ECG shows accentuation of r′ and development of a type 1 ST segment elevation in V₁ and V₂ after ajmaline; QT and QTc are normal.

Figure 4. Genetic analysis of proband 1 and the affected family members

A: Human SCN5A proposed secondary structure showing the location of theE1784K mutation in the C-terminal of the channel. B: PCR-based sequence of SCN5A exon 28 showing wild type (WT) and heterozygous G to A transversion at nucleotide 5350 (arrow) in patient I-2 and II-1. The mutation predicts a substitution of Lys (AAG) for Glu (GAG) at position 1784 (E1784K). C: Sequence analysis of exon 34 of ABCC9 in patient I-1 and II-1, showing the heterozygous C to G transversion at nucleotide 4205 (arrow), predicting a substitution of cysteine (TGC) for serine (TCC) at position 1402 (S1402C).

Figure 5. S1402C mutation in ABCC9

A: Location of S1402C mutation in SUR2A structure within nucleotide binding domain (NBD) 2 is indicated by red dot. W_A and W_B denote the conserved Walker motifs within ABC protein family that are critical for nucleotide binding. B: Alignment of the human SUR2A Ser1402 and surrounding region with orthologs from other species revealed that the mutation located within a highly conserved region. C: Homology structural model of the NBD1/NBD2 heterodimer maps S1402C mutation to the region adjacent to the conserved NBD2 linker and CKC motifs. N- and C-termini of individual NBDs. Yellow denotes α-helix, cyan β-strand, green Walker A or B motif (WA or WB) or linker motif, blue ATP, magenta chemical knockout/gene complementation motif, and red Ser1402.

Figure 6. ABCC9-S1402C mutation reduces sensitivity of ATP-sensitive potassium channel (K_ATP) to ATP

A and B: I_K-ATP traces recorded from excised, inside-out macropatches, elicited by applying ramps from −80 to +80 mV (duration, 3 s), before and after attaining steady-state inhibition with K₂ATP. Currents were normalized to the current recorded at −80 mV under control conditions. C: Concentration-response curves for the effects of K₂ATP on KCNJ11- wild type (WT)/ABCC9-WT and KCNJ11-WT/ABCC9-S1402C mutant channels. Currents were measured at −80 mV. The half-maximal inhibitory concentration (IC₅₀) value of K₂ATP with KCNJ11-WT/ABCC9-WT (13.4 ± 5 μM; n = 6) was significantly higher for the KCNJ11-WT/ABCC9-S1402C mutant channel (8.5 ± 2 mM; n = 6, p < 0.05).

Figure 7. SCN5A-E1784K mutation leads to a loss of function of I_Na,peak, thus inducing a Brugada syndrome phenotype

A: Current-voltage relationship for wild type (WT) (squares, n = 22), E1784K (triangles, n = 23), and E1784K+WT (circles, n = 18). Peak E1784K+WT and E1784K current at −35 mV decreased 30.0% and 61.0% relative to WT, respectively (P <0.05 and P <0.01). B: Voltage-dependent channel inactivation for WT (squares, n = 22), E1784K (triangles, n =17) and E1784K+WT (circles, n = 7). Peak current was normalized to their respective maximum values and plotted against the conditioning potential. Data were fitted by Boltzman function. Steady-state inactivation of E1784K and E1784K+WT was 18.4 mV and 11.0 mV more negative than WT (p < 0.01 and 0.01). C–E: Whole-cell representative peak currents of SCN5A-WT and/or SCN5A-E1784K (panel C) shows the decreased peak I_Na current, proposed to underlie the action potential (AP) changes (panel D) and the electrocardiogram (ECG) manifestation (panel E) of Brugada syndrome (BrS). Endo: endocardial; Epi: epicardial. Traces in panels D and E are diagrammatic.

Figure 8. SCN5A-E1784K mutation leads to a gain of function of I_Na,late, thus inducing a long QT syndrome phenotype

A–B: Bar graph of relative I_Na,late (% of I_Na,peak) and I_Na,late density (pA/pF) among 3 groups. Statistically significant differences (*P<0.05) are observed between E1784K+wild type (WT)/E1784K and WT in both panels (n = 22, 15, 5 for WT, E1784K and E1784K+WT). I_Na,late was 0.14%, 2.01% and 1.86% of I_Na,peak in WT, E1784K and E1784K+WT (n =22, 15 and 5). C–E: Whole-cell late currents of SCN5A-WT and/or SCN5A-E1784K (panel C) showing a larger I_Na,late for the mutant channel and its effect to prolong action potential (AP) duration (panel D) and QT interval (panel E), which cause LQTS-3. ECG: electrocardiogram. I_Na,peak in panel C is normalized to the same level; traces in panels D and E are diagrammatic.